CN100537743C - A kind of beer yeast engineering bacteria and preparation method thereof and application - Google Patents
A kind of beer yeast engineering bacteria and preparation method thereof and application Download PDFInfo
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- CN100537743C CN100537743C CNB2006101137401A CN200610113740A CN100537743C CN 100537743 C CN100537743 C CN 100537743C CN B2006101137401 A CNB2006101137401 A CN B2006101137401A CN 200610113740 A CN200610113740 A CN 200610113740A CN 100537743 C CN100537743 C CN 100537743C
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Abstract
The invention discloses a kind of beer yeast engineering bacteria and preparation method thereof and application.This beer yeast engineering bacteria is with the PEP4 gene disruption in the yeast saccharomyces cerevisiae, and imports the glutathione synthetase gene of yeast saccharomyces cerevisiae, and what obtain does not produce protease A and the high bacterial strain of the described yeast saccharomyces cerevisiae of gsh rate ratio.Described beer yeast engineering bacteria is preferably yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5 CGMCC № .1787.Beer yeast engineering bacteria of the present invention is compared with common saccharomyces cerevisiae (recipient bacterium), and froth stability raises, and glutathione content increases, and its physiology, biochemical characteristic no significant difference.
Description
Technical field
The present invention relates to a kind of beer yeast engineering bacteria and preparation method thereof and application.
Background technology
Draft beer is in storage process, and its froth stability descends gradually, mainly be since in the draft beer residual endotrypsin A to due to the proteic Degradation of beer foam; In addition, the same with ordinary beer, behind the beer packed in transportation and storage process because the compounds such as some trans diene aldehydes that oxygenizement produces, make generation some stale flavors beastly, i.e. Cooked Taste in the beer.Beer foam stability and flavor stability are the important indicator of weighing beer quality, though in recent years by improving processing condition and selecting for use sophisticated equipment etc. can improve the froth stability of beer to a certain extent and reduce the possibility of beer oxidation, but will fundamentally solve draft beer foam and problem of oxidation still needs to utilize the molecular biology breeding technique to make up protease A disappearance, the cereuisiae fermentum industrial strain of homoglutathion content.
Summary of the invention
The purpose of this invention is to provide a kind of beer yeast engineering bacteria and construction process thereof and application.
Beer yeast engineering bacteria provided by the present invention is with the PEP4 gene disruption in the yeast saccharomyces cerevisiae, and imports the glutathione synthetase gene of yeast saccharomyces cerevisiae, and what obtain does not produce protease A and the high bacterial strain of the described yeast saccharomyces cerevisiae of gsh rate ratio.
Described yeast saccharomyces cerevisiae can be yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.597 (available from China Committee for Culture Collection of Microorganisms); It is 5 of D90220 ' end 31-4220 position deoxynucleotide that the nucleotide sequence of described glutathione synthetase gene contains from GenBank Accession Number at least.
Described beer yeast engineering bacteria is preferably yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5CGMCC № .1787.
Described yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5, be preserved in Chinese microorganism strain preservation board of trustee reason person on 08 28th, 2006 and understood common micro-organisms center (abbreviation CGMCC, the address is: No. 13, one in Zhong Guan-cun, Haidian District, BeiJing, China city north), preserving number is CGMCC № .1787.
The cell of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5CGMCC № .1787 is oval, and colony morphology characteristic is bacterium colony projection, smooth, oyster white, neat in edge.30 ℃ of optimum growth temperatures.The growth optimal pH is 5.5.
Second purpose of the present invention provides a kind of method that makes up above-mentioned beer yeast engineering bacteria.
The method of the above-mentioned beer yeast engineering bacteria of structure provided by the present invention, be that following recombinant fragment is imported yeast saccharomyces cerevisiae, obtain do not produce protease A and the high bacterial strain of the described yeast saccharomyces cerevisiae of gsh rate ratio is beer yeast engineering bacteria: the length that described recombinant fragment comprises glutathione synthetase gene (GSH1), be selected from 5 ' terminal sequence of PEP4 gene is at least the nucleotide fragments 2 that the nucleotide fragments 1 of 45bp, the length that is selected from 3 ' terminal sequence of PEP4 gene are at least 45bp;
Described glutathione synthetase gene (GSH1) is between described nucleotide fragments 1 and nucleotide fragments 2;
It is 5 of D90220 ' end 31-4220 position deoxynucleotide that the nucleotide sequence of described glutathione synthetase gene contains from GenBank Accession Number at least.
5 ' terminal sequence of described PEP4 gene is to be 5 of M13358 M13632 ' end 1-300 position deoxynucleotide from GenBank Accession Number, and 3 ' terminal sequence of described PEP4 gene is to be 5 of M13358 M13632 ' end 1987-2146 position deoxynucleotide from GenBank AccessionNumber.
In the described method, described nucleotide fragments 1 is positioned at 3 of described glutathione synthetase gene ' end, and described nucleotide fragments 2 is positioned at 5 of described glutathione synthetase gene ' end;
The encoding sequence of described glutathione synthetase gene is to be 5 of D90220 ' end 31-4220 position deoxynucleotide from GenBank Accession Number.
In the described method, it is 5 of M13358M13632 ' end 46-247bp position deoxynucleoside acid sequence that described nucleotide fragments 1 is preferably GenBank Accession Number; It is 5 of M13358 M13632 ' end 1987-2105bp position deoxynucleoside acid sequence that described nucleotide fragments 2 is preferably GenBankAccession Number.
In the described method, described recombinant fragment also comprises copper resistant gene C UP1; Described copper resistant gene C UP1 is between described glutathione synthetase gene and described nucleotide fragments 1;
The encoding sequence of described copper resistant gene C UP1 is for from GenBank Accession Number being 5 of K02204M64045 ' end 1098-2010 position deoxynucleotide.
In the described recombinant fragment, 5 of described copper resistant gene C UP1 ' end is positioned at 3 of described nucleotide fragments 1 ' end, 3 of described copper resistant gene C UP1 ' end is positioned at 3 of described glutathione synthetase gene ' end, and 5 of glutathione synthetase gene ' end is positioned at 5 of described nucleotide fragments 2 ' end.
Described recombinant fragment is the fragment of cutting the 7.0kb of pPCG1 acquisition as shown in Figure 6 with AatII and PstI enzyme.
Beer yeast engineering bacteria of the present invention is with the PEP4 gene disruption in the common saccharomyces cerevisiae, and a copy obtains and simultaneously copper resistant gene and glutathione synthetase gene have all been increased.Prove by genetic stability experiment, the experiment of simulation beer fermentation and lab scale experiment fermenting experiment, beer yeast engineering bacteria of the present invention is compared with common saccharomyces cerevisiae (recipient bacterium), froth stability raises, and glutathione content increases, and its physiology, biochemical characteristic no significant difference; Engineering bacteria does not have particular requirement to fermentation equipment and condition, and the equipment and the condition of general draft beer factory all can be used.
Description of drawings
Fig. 1 is the PCR product electrophorogram of PEP4
Fig. 2 is the physical map of recombinant plasmid pYPEP
Fig. 3 cuts the checking electrophorogram for the enzyme of recombinant plasmid pYPEP
Fig. 4 is the structure schematic flow sheet of recombinant plasmid pPEP-1
Fig. 5 cuts the checking electrophorogram for the enzyme of recombinant plasmid pPEP-1
Fig. 6 is the structure schematic flow sheet of recombinant plasmid pPCG1
Fig. 7 cuts the checking electrophorogram for the enzyme of recombinant plasmid pPCG1
Fig. 8 is the structure synoptic diagram of yeast saccharomyces cerevisiae ZWYT-5CGMCC № .1787
Fig. 9 is the copper resistant detected result of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5 CGMCC № .1787
Figure 10 is the PCR Molecular Identification result of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5 CGMCC № .1787
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Used percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
The acquisition and the application experiment thereof of the industrial beer yeast engineering bacteria of embodiment 1, protease A disappearance homoglutathion content
Owing to use the gene constructed engineering bacteria that derives from non-industrial microorganism whether suitable for drinking, can also to need to be accepted facts have proved for a long time by the human consumer, and cereuisiae fermentum itself just contains the glutathione synthetase gene, so adopt gene glutathione synthetase gene (is 5 of D90220 ' end 31-4220 position deoxynucleotide from GenBank Accession Number) to be inserted among the endotrypsin A gene PEP4 (is 5 of M13358 M13632 ' end 247-1987 position deoxynucleotide from GenBank Accession Number) from clone's mode, destroyed and the glutathione synthetase gene has increased the engineering bacteria of a copy with regard to having made up the PEP4 gene like this.This project bacterium makes Foam Stability of Unpasteurized Beer improve because of protease A disappearance, and the increase of glutathione content can make the beer refreshing time lengthening.
One, the acquisition of the industrial beer yeast engineering bacteria of protease A disappearance homoglutathion content
1, the screening of the industrial saccharomyces cerevisiae that copper resistant is low
Industrial saccharomyces cerevisiae is generally on the low side to the resistance of copper, therefore, can effectively screen yeast transformant according to the raising of copper resistant, and copper resistant generally is used as the selective marker of industrial cereuisiae fermentum.Measure by copper resistant, screen three strain yeast and containing 3.0mmol/L CuSO
4The YEPD flat board on almost can not grow, determine the recipient bacterium as next step transformation experiment with wherein a strain AS2.597 (available from China Committee for Culture Collection of Microorganisms).
2, the structure of recombinant expression plasmid carrier pPCG1 and enzyme are cut checking
1) structure and the enzyme that contains the recombinant plasmid pYPEP of PEP4 gene cut checking
The acquisition of the PEP4 gene of A, coding endotrypsin A
PEP4 gene order according to the cereuisiae fermentum of having reported designs corresponding primer, introduces BamH I and Sal I restriction enzyme site (shown in the underscore) respectively, carries out the pcr amplification of PEP4 gene.Primer sequence is:
Upstream primer F1:5 ' GCAGGATCCCGTTTTGATACGATTTAC 3 ' (BamH I)
Downstream primer R1:5 ' CAGGTCGACTGGGCAGCAGCATAGAAC 3 ' (Sal I)
As template, carry out pcr amplification with total DNA of industrial Yeast strain of beer AS2.597, in 50 μ l PCR reaction systems, contain 25 μ l Taq enzyme Mix, each 2 μ L of 10 μ mol/L primers F 1 and R1, template 10 μ l add sterilized water 11 μ l and are adjusted to 50 μ l.Response procedures is: 94 ℃ earlier, and sex change 5min; 94 ℃ of sex change 40s then, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, totally 30 circulations; Last 72 ℃ of extension 15min extend fully product.The PCR product is through 0.8% agarose gel electrophoresis, can see the specific band of a treaty 2.0kb, its size and expection suitable (Fig. 1), show that through order-checking this fragment has the encoding sequence of PEP4 gene (GenBankAccession Number is that M13358 M13632 is from 5 ' end 46-2105 position deoxynucleoside acid sequence), wherein swimming lane 1 is Marker among Fig. 1, and swimming lane 2 is the PCR product of PEP4.
Structure and the enzyme of B, recombinant vectors pYPEP are cut checking
Recycling step 1) the PCR product of Huo Deing, behind BamHI and Sal I double digestion, insert carrier YIp5 (GenBank number: between BamHI L09157) and the Sal I restriction enzyme site, and it is cut with the BamHI enzyme respectively, BamH and SalI double digestion, the EcoRI enzyme is cut, the EcoRV enzyme is cut, the HindIII enzyme is cut, SacI and XbaI double digestion, the PstI enzyme is cut and is verified, the result shows that the BamHI enzyme cuts the band (swimming lane 2 among Fig. 3) that obtains 7331bp, BamHI and SalI double digestion obtain 2066 and the band (swimming lane 3 among Fig. 3) of 5265bp, the EcoRI enzyme is cut and is obtained 982 and the band (swimming lane 4 among Fig. 3) of 6349bp, the EcoRV enzyme is cut and is obtained 1649,1800 and the band (swimming lane 5 among Fig. 3) of 3882bp, the HindIII enzyme is cut and is obtained 1440 and the band (swimming lane 6 among Fig. 3) of 5891bp, SacI and XbaI double digestion obtain 1455 and the band (swimming lane 7 among Fig. 3) of 5591bp, the PstI enzyme is cut and is obtained 3151 and the band (swimming lane 8 among Fig. 3) of 4180bp, enzyme is cut the recombinant vectors called after pYPEP (Fig. 2) that evaluation shows the correct encoding sequence that contains the PEP4 gene (GenBank Accession Number is that M13358 M13632 is from 5 ' end 46-2105 position deoxynucleoside acid sequence), and wherein swimming lane 1 is a molecular weight standard among Fig. 3.
2) structure of recombinant clone plasmid vector pPEP-1 and enzyme are cut checking
The structure of recombinant clone plasmid vector pPEP-1 as shown in Figure 4, wherein, pICG is that (GenBank number: the fragment of the 12bp L14758) between BamHI and the SalI site replaces with the following fragment gained of polyphone successively: 5 ' terminal sequence of acetolactate synthase gene ILV2 (is 5 of X02549 ' end 442-707 position deoxynucleotide from GenBank AccessionNumber) with the YEP352 plasmid, CUP1 gene (is 5 of K02204M64045 ' end 1098-2010 position deoxynucleotide from GenBankAccession Number), GSH1 gene (is 5 of D90220 ' end 31-4220 position deoxynucleotide from GenBank Accession Number), 3 ' terminal sequence of ILV2 (is 5 of X02549 ' end 2876-3371 position deoxynucleotide from GenBank Accession Number); Wherein, YEP352 plasmid BamHI site end is connected with 5 ' end of 5 ' terminal sequence of acetolactate synthase gene ILV2,3 of 5 ' terminal sequence of acetolactate synthase gene ILV2 ' end is connected with 5 of CUP1 gene ' end, 3 of CUP1 gene ' end is connected with 3 of GSH1 gene ' end, 5 of GSH1 gene ' end is connected with 5 ' end of 3 ' terminal sequence of ILV2, and 3 of 3 ' terminal sequence of ILV2 ' end is connected with YEP352 plasmid SalI site end.
With SacI and XbaI enzyme cutting plasmid pICG, (5 ' end length is the sequence of 265bp to obtain containing part ILV2 sequence, be 5 of X02549 ' end 442-707 position deoxynucleotide promptly from GenBank Accession Number) and part copper resistant gene CUP1 (5 ' end length is the sequence of 208bp, be 5 of K02204 M64045 ' end 1098-1305 position deoxynucleotide promptly from GenBank Accession Number) dna fragmentation, with its called after ILV+CUP, its length is 473bp, this fragment is inserted between the SacI and XbaI enzyme cutting site of PEP4 gene of pYPEP, the segmental two ends of ILV+CUP are connected with 3 ' terminal sequence (is 5 of M13358 M13632 ' end 1987-2105 position deoxynucleoside acid sequence from GENBANK Accession Number) with 5 ' terminal sequence (is 5 of M13358 M13632 ' end 46-247 position deoxynucleoside acid sequence from GENBANK AccessionNumber) of PEP4 respectively, obtain recombinant vectors, it is cut with the EcoRV enzyme, the BglII enzyme is cut, the BamHI+SalI double digestion, the PstI enzyme is cut the evaluation recombinant plasmid, the result as shown in Figure 5, the result shows, the EcoRV enzyme is cut and is obtained 797,1393 and the product (swimming lane 1) of 3882bp, the BglII enzyme is cut the product (swimming lane 2) that obtains 6072bp, the BamHI+SalI double digestion obtains 807 and the product (swimming lane 3) of 5265bp, the PstI enzyme is cut the product (swimming lane 4) that obtains 2921bp and 3151bp, swimming lane 5 is a molecular weight standard among Fig. 5, and evaluation is shown correct recombinant vectors called after pPEP--1 (making up schematic flow sheet as shown in Figure 4).
3) structure of recombinant expression plasmid carrier pPCG1 and enzyme are cut checking (the building process synoptic diagram as shown in Figure 6)
The building process of the used plasmid pYCUP of structure of recombinant expression plasmid carrier pPCG1 is: 5 ' end interpolation BglII and 3 ' is held the copper resistant gene C UP1 fragment (is 5 of K02204 M64045 ' end 1098-2010 position deoxynucleoside acid sequence from GenBank AccessionNumber) of adding the SalI recognition site, and linking to each other with pBluescript II SK (-) plasmid of cutting through the HincII enzyme (available from Stratagene company) obtains the pMCUP plasmid, KpnI one end of BglII site on pBluescript M13 among the CUP1 wherein, the SalI site is at the HincII of pBluescript II SK (-) one end among the CUP1; Cutting CUP1 fragment that the pMCUP plasmid obtains with KpnI and SalI enzyme then is connected with the YEP352 plasmid of KpnI and SalI double digestion and is built into the pYCUP plasmid.
The building process of the used plasmid pGF-2 of structure of recombinant expression plasmid carrier pPCG1 is: the glutathione synthetase gene GSH1 fragment (is 5 of D90220 ' end 31-4220 position deoxynucleoside acid sequence from GenBank AccessionNumber) of 5 ' end being added SacI and 3 ' end interpolation SalI site is connected with the YEP352 plasmid of cutting through the SmaI enzyme, wherein among the GSH1 SacI site at the SmaI of YEP352 plasmid one end, the SalI site is built into the pGF-2 plasmid at the KpnI of YEP352 plasmid one end.
With BglII and SalI digested plasmid pYCUP, obtain CUP1 fragment (is 5 of K02204 M64045 ' end 1098-2010 position deoxynucleoside acid sequence from GenBank Accession Number);
With SalI and XbaI enzyme cutting plasmid pGF-2, obtain glutathione synthetase gene (GSH1) fragment (is 5 of D90220 ' end 31-4220 position deoxynucleoside acid sequence from GenBank Accession Number) of 4189bp;
With BglII and XbaI enzyme cutting plasmid pPEP-1, obtain the fragment of 5858pb, this fragment is connected with glutathione synthetase gene (GSH1) fragment with above-mentioned copper resistant gene (CUP1) fragment that obtains, obtain recombinant plasmid pPCG1 (Fig. 6), with this recombinant plasmid pPCG1 AatII, PstI, EcoRI, BglII, SalI enzyme respectively cuts evaluation, the result as shown in Figure 7, the AatII enzyme is cut the fragment (swimming lane 1) that recombinant plasmid obtains 10991bp, the PstI enzyme is cut recombinant plasmid and is obtained 3151 and the fragment (swimming lane 2) of 7840bp, the EcoRI enzyme is cut recombinant plasmid and is obtained 2477,3310 and the fragment (swimming lane 3) of 5204bp, the BglII enzyme is cut recombinant plasmid and is obtained 507,930 and the fragment (swimming lane 4) of 9554bp, the SalI enzyme is cut recombinant plasmid and is obtained 4333 and the fragment (swimming lane 5) of 6658bp, and swimming lane 6 is a molecular weight standard among Fig. 7.Enzyme is cut evaluation and is shown that this recombinant plasmid pPCG1 structure is correct.
3, the acquisition (the building process synoptic diagram as shown in Figure 8) of industrial saccharomyces cerevisiae engineering bacteria-yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5CGMCC № .1787
Cut the dna fragmentation that recombinant plasmid pPCG1 obtains a 7.0kb size with AatII and PstI enzyme.This fragment contains copper resistant gene (CUP1) and glutathione synthetase gene (GSH1), and two ends are 5 of PEP4 gene ' end and 3 ' terminal sequence.This dna fragmentation is transformed industrial saccharomyces cerevisiae AS2.597 with Lithium Acetate intact cell method for transformation, make the PEP4 on this fragment and the AS2.597 genome that homologous recombination take place, the result, copper resistant gene and gsh gene are integrated on the karyomit(e).Like this, in restructuring yeast strains, the PEP4 gene is destroyed, and while copper resistant gene and glutathione synthetase gene have all increased a copy.With restructuring yeast strains called after yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5 that obtains, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 08 28th, 2006, the address is: No. 13, one in Zhong Guan-cun, Haidian District, BeiJing, China city north), preserving number is CGMCC № .1787.
The cell of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5 CGMCC № .1787 is oval, and after cultivating 2 days on the YEPD substratum, its colony morphology characteristic is bacterium colony projection, smooth, oyster white, neat in edge.30 ℃ of optimum growth temperatures.The growth optimal pH is 5.5.
Two, the checking of industrial saccharomyces cerevisiae engineering bacteria-yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5CGMCC № .1787
1, the molecule of engineering bacteria checking
1) Detection of Stability of engineering bacteria
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5 CGMCC № .1787 there is being selective pressure (YEPD+5mmol/LCuSO respectively
4) and do not have in the liquid nutrient medium of selective pressure (YEPD) continuous passage and cultivated 5 days, every day, sampling detected the loss situation of the dna fragmentation that inserts, and concrete grammar is:
Extracting yeast chromosomal dna, is template with it, verifies (with following " 3) pcr amplification checking ") with PCR method.
The result shows no matter having under the selective pressure condition, and still under no selective pressure condition, behind the cultured continuously 120h, 100% engineering bacteria cell has the insertion dna fragmentation, does not lose phenomenon.
2) copper resistant of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5CGMCC № .1787 detects
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5 CGMCC № .1787 and recipient bacterium yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.597 (available from China Committee for Culture Collection of Microorganisms) be seeded in respectively contain 3mmol/L, 5mmol/L, on the YEPD flat board of 7mmol/L CuSO4, after cultivating 3 days under 30 ℃ of conditions, observations, the result as shown in Figure 9, show that recipient bacterium yeast saccharomyces cerevisiae (Saccharomycescerevisiae) AS2.597 is containing on the YEPD flat board of the above CuSO4 of 3mmol/L and can not grow, and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5 CGMCC № .1787 is at 3mmol/L, 5mmol/L, all can grow on the YEPD flat board of 7mmol/L CuSO4; Among Fig. 9, each culture dish left side five bacterium colonies of several first row are recipient bacterium AS2.597, and all the other 7 classify yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5CGMCC № .1787 as.
3) pcr amplification checking
Be template with the genomic dna of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5 CGMCC № .1787 and the genomic dna of recipient bacterium yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.597 respectively, various combination (PEP4-A and CUP1-B with the primer described in the table 1, GSH1-B and PEP4-D, PEP4-A and PEP4-D) carry out pcr amplification, amplified production carries out detected through gel electrophoresis, the result as shown in figure 10, the result shows, the genomic dna of recipient bacterium yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.597 is with PEP4-A and CUP1-B combination of primers amplification (swimming lane 5 among Figure 10) and GSH1-B and PEP4-D combination of primers amplification (swimming lane 6 among Figure 10) fragment that all do not increase, and is 2.0kb (PEP4 gene (is that 5 of M13358M13632 ' holds 46-2105 position deoxynucleotide from GENBANK Accession Number) with the PCR product that PEP4-A and PEP4-D combination of primers (swimming lane 4 among Figure 10) increase; The genomic dna of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5CGMCC № .1787 obtains amplified production (5 ' terminal sequence of PEP4 (is 5 of M13358 M13632 ' end 46-247 position deoxynucleoside acid sequence from GENBANK Accession Number) of 1.5kb with PEP4-A and CUP1-B combination of primers amplification (swimming lane 1 among Figure 10), 5 ' terminal sequence of ILV2 gene (is 5 of X02549 ' end 442-707 position deoxynucleotide from GenBank Accession Number) and CUP1 gene (is 5 of K02204 M64045 ' end 1098-2010 position deoxynucleotide from GenBank Accession Number)), obtain amplified production (the GSH1 gene (is 5 of D90220 ' end 31-4220 position deoxynucleotide from GenBank Accession Number) of 4.3kb with GSH1-B and PEP4-D combination of primers amplification (swimming lane 2 among Figure 10), PEP4 gene 3 ' terminal sequence (is 5 of M13358M13632 ' end 1987-2105 position deoxynucleoside acid sequence from GENBANK Accession Number)), the PCR product size that increases with PEP4-A and PEP4-D combination of primers (swimming lane 3 among Figure 10) is 5.7kb (5 ' terminal sequence of PEP4 (is 5 of M13358 M13632 ' end 46-247 position deoxynucleoside acid sequence from GENBANKAccession Number), 5 ' terminal sequence of ILV2 gene (is 5 of X02549 ' end 442-707 position deoxynucleotide from GenBank Accession Number), CUP1 gene (is 5 of K02204 M64045 ' end 1098-2010 position deoxynucleotide from GenBank Accession Number), GSH1 gene (is 5 of D90220 ' end 31-4220 position deoxynucleotide from GenBank Accession Number), PEP4 gene 3 ' terminal sequence (is 5 of M13358 M13632 ' end 1987-2105 position deoxynucleoside acid sequence from GENBANK AccessionNumber)), this result is consistent with the expection size, illustrate that yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 is with the PEP4 gene disruption among recipient bacterium yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.597, and CUP1 and GSH1 gene integration to PEP4 gene position are obtained, can express CUP1 and GSH1 gene and do not express the PEP4 gene.Swimming lane 7 is a molecular weight standard among Figure 10.
Table 1.PCR amplification checking primer
| Primer | Sequence 5 ' → 3 ' |
| CUP1-B GSH1-B PEP4-A PEP4-D | ATCTGTTGTACTATCCGCTT TACA AGATCTAACAGGAGCA CGTTTTGATACGATTTAC TGGGCAGCAGCATAGAAC |
2, endotrypsin A activity and glutathione content are measured
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.597 and yeast saccharomyces cerevisiae ZWYT-5CGMCC № .1787 are respectively after slant activation, insert at first respectively in the 10mlYEPD liquid, 30 ℃ of shaking tables (200rpm) were cultivated after 18 hours, inoculum size by 5% is connected in the 100mlYEPD liquid, under similarity condition, cultivate after 72 hours, carry out endotrypsin A determination of activity and glutathione content mensuration in thalline and the fermented liquid.
Endotrypsin A activity determination method in the thalline:
(1) after yeast is cultivated in the YEPD substratum, centrifugal collection thalline;
(2) wash once with the potassium phosphate buffer of 0.1M pH7.0, be suspended in the 0.2M potassium phosphate buffer of 1/10th volumes (former culture volume) then again, final pH 7.0;
When (3) measuring enzyme and live, with ultrasonic cell disruptor with cytoclasis (in 5 seconds once, one minute at interval, totally 20 times), centrifugal 5 minutes of 5000rpm, supernatant liquor (enzyme liquid) is standby.
(4) 1% casein solution (pH2.2) are incubated 20min with the enzyme liquid that step (3) obtains in 37 ℃, after adding 10% Tricholroacetic Acid 2ml termination reaction 10min, the centrifugal 15min of 15000g, supernatant liquor Bradford method (0.1ml supernatant liquor and the reaction of 3ml protein reagent) is the mensuration optical density value (595nm).Blank is: the enzyme in the reaction solution is earlier with 10% Tricholroacetic Acid deactivation 15min.
Endotrypsin A activity unit: the optical density value (OD that in above-mentioned Bradford method, 0.1ml bovine serum albumin (0.1mg/ml) and the reaction of 3ml protein reagent is produced
595) be defined as an enzyme activity unit.
Endotrypsin A activity determination method in the fermented liquid:
(1) fermented liquid 100ml is in 4 ℃ of following 50% ammonium sulfate saturation ratios 2h that saltout, the centrifugal 20min of 20000g, and precipitation is the protease A crude extract with water dissolution and constant volume to 1ml.
(2) measuring method is with above-mentioned " (4) "
Gsh measuring method in the thalline:
(1) get fermented liquid 5ml, centrifugal 5 minutes of 4000rpm adds 5ml distilled water wash precipitation 2 times, adds 1ml distilled water ,-20 ℃ of freezing spending the night;
(2) boil 5 minutes in the boiling water bath, add 4ml distilled water, the centrifuging and taking supernatant liquor is measured; 80 ℃ of thalline are dried to constant weight and weigh.
(3) in colorimetric cylinder, add following solution successively: 1.5ml 0.15N NaOH; 0.5ml liquid to be measured (0.5ml distilled water is blank); 0.5ml 3% formaldehyde; 2.5ml the DTNB developer, mixing, the 412nm colorimetric was reacted 5 minutes in 25 ℃ of water-baths down;
(4) GSH (mg/g stem cell)=OD
412÷ 3.7236 ÷ stem cells gram number
Gsh measuring method in the fermented liquid:
(1) in colorimetric cylinder, adds following solution successively: 1.5ml 0.15N NaOH; 1ml fermented liquid (1ml distilled water is blank); 0.5ml 3% formaldehyde; 2.5ml the DTNB developer, mixing, the 412nm colorimetric was reacted 5 minutes in 25 ℃ of water-baths down;
(2)GSH(mg/100ml)＝OD
412×100÷3.7236
The protease A determination of activity is the result show, detects in yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 less than protease A activity (table 2), and just proof PEP4 gene in yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 is destroyed for this.
The glutathione content measurement result shows (table 2), no matter is in the somatic cells, or in the fermented liquid, the glutathione content of yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 all is improved.
The protease A activity of table 2. recipient bacterium and yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 and glutathione content are relatively
3, laboratory ferment experimental result
Yeast saccharomyces cerevisiae AS2.597 and yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 are inserted 5ml malt extract medium (pol is the wort of 10 ° of P) respectively, cultivated 12 hours for 25 ℃, inoculum size with 10% (v/v) is inoculated in the 10ml malt extract medium, cultivated 36 hours for 25 ℃, all be connected in the triangular flask that contains the 200ml wort, 10 ℃ leave standstill cultivation 15 days, (fermented liquid) method detection glutathione content and the protease A activity according to step 2 of taking a sample every three days.
The result shows among the yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 does not have the protease A activity in the fermented liquid, and the content of gsh is than the height (table 3) in the recipient bacterium fermented liquid, and this explanation GSH1 gene in engineering bacteria has obtained high expression level and the PEP4 gene successfully knocks out.
The glutathione content of table 3. recipient bacterium and yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 relatively
4, lab scale (hectolitre) fermenting experiment
1) beer fermentation carries out as follows:
Recipient bacterium AS2.597 or yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 after inclined-plane and the liquid activation are seeded in the 70L wort (pol is 10 ° of P) by 10% respectively, 10 ℃ fermented 12 days, sealed cans, 0 ℃ of cold storage in the time of 16 days was filtered with canned in the time of the 20th day.
2) glutathione content and endotrypsin A are active detects
After beer fermentation finishes, detect glutathione content in the active and fermented liquid of endotrypsin A in the fermented liquid according to the method for step 2, the result shows in the yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 fermented liquid and does not detect endotrypsin A activity; Glutathione content is 3.1mg/100ml in the beer of yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 fermentation, glutathione content is 2.2mg/100ml in the beer of recipient bacterium fermentation, glutathione content is 1.4 times of recipient bacterium fermentation beer in the beer of yeast saccharomyces cerevisiae ZWYT-5CGMCC № .1787 fermentation, explanation protease A gene in yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 cell is deleted, and the GSH1 gene has obtained high expression level.
3) detection of other indexs after the fermentation ends
The hectolitre experiment detects diacetyl content and fermentation degree in back storage phase sampling.
The diacetyl content measuring method is as described below:
(1) the di-acetyl distiller is installed, bottle takes place in heating steam, makes water to boiling, and after the logical vapour preheating, puts the 25ml volumetric flask in the condenser end opening, adds the ice bath cooling.
(2) add 2～4 defoamers (silicone antifoam agent) in the 100ml graduated cylinder, reinject without degasification be chilled to wine liquid 100ml about 5 ℃ in advance, add in the distiller of preheating in the step (1) capping plug rapidly.
(3) water seals then, distills, and during near 25ml, takes off volumetric flask until distillate, and water is settled to the 25ml scale, shakes up.
(4) draw distillate 10ml respectively in two colorimetric cylinders, and in first arm, add the 0.5ml o-phenylenediamine solution, after not adding (doing blank) in second arm and fully shaking up, place the dark place to place 20～30min simultaneously.
(5) in first colorimetric cylinder, add 2ml hydrochloric acid soln (4mol/L) then, in second arm, add the 2.5ml hydrochloric acid soln, behind the mixing, survey its absorbancy in 335nm in the 20min.
(6) X=A
335* 2.4, in the formula: the content of X-di-acetyl (mg/L); A
335Be the absorbancy under the 335nm wavelength; 2.4 be the reduction factor of absorbancy and diacetyl content.
The fermentation degree measuring method:
Get fermented liquid, the elimination yeast, the low baking temperature heating evaporation to original volume 1/3 to remove ethanol.Proportion when adding water and recovering behind the original volume with 20 ℃ of pycnometric determinations is calculated as follows attenuation real degree.
In the formula: wort concentration (° P) before w-fermentation
w
1The fermented liquid concentration (° P) behind the alcohol is got rid of in-fermentation back
w
r-attenuation real degree (%)
The result shows basic identical (all about 0.03mg/L of diacetyl content of the main fermentation index of recipient bacterium and engineering bacteria; Fermentation degree all about 65%), illustrate that the integration on the karyomit(e) does not change other characteristics of zymic.
4) beer foam performance detects
After beer after canned placed 20 ℃ of constant temperature water bath 30min, survey bubble with Huffman foaming test apparatus (NIBEM method) and hold the time.The bubble time of holding of engineering bacteria yeast saccharomyces cerevisiae ZWYT-5 CGMCC № .1787 brewing beer is 230s, holds long 20s of time than the bubble of recipient bacterium AS2.597 brewing beer, illustrates that the foaming properties of engineering bacteria brewing beer is better than recipient bacterium.
Claims (8)
1, a kind of beer yeast engineering bacteria, be with the PEP4 gene generation homologous recombination in a recombination sequence and the yeast saccharomyces cerevisiae, make the PEP4 gene disruption in the yeast saccharomyces cerevisiae, and import the glutathione synthetase gene of yeast saccharomyces cerevisiae, what obtain does not produce protease A and the high bacterial strain of the described yeast saccharomyces cerevisiae of gsh rate ratio; Described recombination sequence comprises 5 ' end 31-4220 position deoxynucleoside acid sequence of the GenBank Accession Number D90220 of glutathione synthetase gene, be connected with copper resistant gene C UP1 at 3 of above-mentioned glutathione synthetase gene ' end, be connected with 5 ' end 1-300 position deoxynucleoside acid sequence of the GenBank Accession Number M13358 M13632 of PEP4 gene on the copper resistant gene C UP1, be connected with 5 ' end 1987-2146 position deoxynucleoside acid sequence of the GenBankAccession Number M13358 M13632 of PEP4 gene at 5 of above-mentioned glutathione synthetase gene ' end.
2, beer yeast engineering bacteria according to claim 1 is characterized in that: described yeast saccharomyces cerevisiae is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.597.
3, beer yeast engineering bacteria according to claim 1 and 2, it is characterized in that: in the described recombination sequence, be connected with 5 ' end 46-247 position deoxynucleoside acid sequence of the GenBank Accession Number M13358 M13632 of PEP4 gene on the copper resistant gene C UP1,5 of glutathione synthetase gene ' end is connected with 5 ' end 1987-2105 position deoxynucleoside acid sequence of the GenBank Accession Number M13358 M13632 of PEP4 gene.
4, beer yeast engineering bacteria according to claim 2 is characterized in that: described beer yeast engineering bacteria is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZWYT-5 CGMCC № .1787.
5, a kind of method that makes up the described beer yeast engineering bacteria of claim 1, be that following recombination sequence is imported yeast saccharomyces cerevisiae, obtain do not produce protease A and the high bacterial strain of the described yeast saccharomyces cerevisiae of gsh rate ratio is beer yeast engineering bacteria: described recombination sequence comprises 5 ' end 31-4220 position deoxynucleoside acid sequence of the GenBank Accession NumberD90220 of glutathione synthetase gene, be connected with copper resistant gene C UP1 at 3 of above-mentioned glutathione synthetase gene ' end, be connected with 5 ' end 1-300 position deoxynucleoside acid sequence of the GenBank AccessionNumber M13358 M13632 of PEP4 gene on the copper resistant gene C UP1, be connected with 5 ' end 1987-2146 position deoxynucleoside acid sequence of the GenBank Accession Number M13358 M13632 of PEP4 gene at 5 of above-mentioned glutathione synthetase gene ' end.
6, method according to claim 5, it is characterized in that: in the described recombination sequence, be connected with 5 ' end 46-247 position deoxynucleoside acid sequence of the GenBank Accession Number M13358 M13632 of PEP4 gene on the copper resistant gene C UP1,5 of glutathione synthetase gene ' end is connected with 5 ' end 1987-2105 position deoxynucleoside acid sequence of the GenBankAccession Number M13358 M13632 of PEP4 gene.
7, according to claim 5 or 6 described methods, it is characterized in that: described copper resistant gene C UP1 is 5 of a GenBankAccession Number K02204 M64045 ' end 1098-2010 position deoxynucleoside acid sequence.
8, the application of the arbitrary described beer yeast engineering bacteria of claim 1-4 in the preparation draft beer.
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| CN104046572B (en) * | 2014-03-21 | 2017-04-05 | 天津科技大学 | One plant of saccharomyces cerevisiae that can reduce biogenic amine in yellow rice wine and its construction method and application |
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